US10802017B2 - Bioanalysis device and biomolecule analyzer - Google Patents

Bioanalysis device and biomolecule analyzer Download PDF

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US10802017B2
US10802017B2 US14/768,771 US201414768771A US10802017B2 US 10802017 B2 US10802017 B2 US 10802017B2 US 201414768771 A US201414768771 A US 201414768771A US 10802017 B2 US10802017 B2 US 10802017B2
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fine particles
solution
magnetic fine
magnetic
magnetic field
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US20160003814A1 (en
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Koshin Hamasaki
Toshiro Saito
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Hitachi High Tech Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/72—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables
    • G01N27/74—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables of fluids
    • G01N27/745—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables of fluids for detecting magnetic beads used in biochemical assays

Definitions

  • the present invention relates to a biomolecule analysis device, and to a biomolecule analyzer using same.
  • Cancer markers are secretory biogenic factors of cancer-cell origin, and increase with cancer progression before they appear in blood or urine.
  • proteins such as hormones and cytokines, and nucleic acids such as microRNAs. These cancer markers occur in very small concentrations in early stages of cancer, and cannot be easily detected. Detection of cancer markers with inherently low expression levels is also difficult.
  • Immunoassays using antibodies have become a mainstream method of high-sensitive cancer marker detection. Techniques such as ELISA and nanoparticle assay are known examples of such a method.
  • Non Patent Literature 1 Recent years have seen more sensitive immunoassays, such as the development of a digital ELISA that enables detection of single molecules.
  • the amount of blood that can be collected from patients is limited, and trace amounts of cancer marker in blood need to be captured for detection in as many numbers as possible.
  • cancer markers are contained in a concentration range of 10 ⁇ 16 to 10 ⁇ 12 M in early stages of cancer, and the detection requires the sensitivity to quantify about 3000 target molecules contained in 50 ⁇ l of a sample. There accordingly is a need for a ultrasensitive detector that enables detection of such low concentrations of cancer markers.
  • Non Patent Literature 1 Rissin D M et al, Nature Biotechnology, June 28(6); p. 595-599 (2010)
  • the present invention relates to a method for quantifying trace amounts of biomolecules, to a structure of a quantification device, and to an analyzer configuration.
  • Efficient capturing of trace amounts of biomolecules suspended in a solution typically requires increasing the frequency by which the target biomolecules collide with the molecules used to capture the target.
  • the present inventors have devised a method that makes use of microscopic magnetic fine particles of sizes no greater than 1 micron as the capture molecules. With the microscopic magnetic fine particles, the surface area per number of particles can be increased, and the molecular mobility can be improved for more efficient reaction with the target biomolecules.
  • the method enables improving the capture efficiency, and increasing the density of immobilizable fine particles in a certain area by making the magnetic fine particles smaller. This is advantageous in terms of the sensitivity and speed of detection.
  • smaller magnetic fine particles have low magnetic susceptibility, and do not easily magnetize.
  • Spreading small magnetic fine particles over a flat substrate thus requires a strong magnetic field and a long magnetization time.
  • the magnetic fine particles have been shown to form beads-like aggregates along the magnetic flux, and become irregularly shaped as such a mass of particles combines with a flat particle layer. This phenomenon is commonly seen particularly at high magnetic fine particle concentrations.
  • a method is used in which a solution containing microscopic magnetic fine particles is confined between flat substrates of high wettability in as thin a vertical thickness as possible, and a magnetic field is applied from the side of one of the flat substrates to attract the magnetic fine particles.
  • a device is used in which magnetic fine particles are flowed into a gap of a certain thickness created between a pair of highly wettable flat substrates, and a magnetic field is applied from the side of one of the flat substrates to immobilize the magnetic fine particles on the substrate.
  • the magnetic fine particles can be evenly immobilized in the form of a film on the substrate surface in a dispersion state. This makes it easier to place the focal point on the fluorescent dye on the substrate, and blocking of excitation light due to trapping of the fluorescent dye by the magnetic fine particles can be prevented.
  • FIG. 1 is a diagram explaining the general principle according to Example of the invention.
  • FIG. 2 is a diagram explaining the structure and the principle of the device of Example of the invention.
  • FIG. 3 is a diagram explaining a method for examining the principle according to Example of the invention.
  • FIG. 4 is a graph explaining the result of principle examination according to Example of the invention.
  • FIG. 5 is a graph explaining the result of principle examination according to Example of the invention.
  • FIG. 6 is a diagram explaining the result of principle examination according to Example of the invention.
  • FIG. 7 is a diagram explaining the result of principle examination according to Example of the invention.
  • FIG. 8 is a diagram explaining the device structure of Example of the invention.
  • FIG. 9 is a diagram explaining an example of the antigen molecule capturing method and the fluorescence labeling method according to Example of the invention.
  • FIG. 10 is a diagram explaining an example of the nucleic acid molecule capturing method and the fluorescence labeling method according to Example of the invention.
  • FIG. 11 is a diagram explaining an example of the configuration of the biomolecule analyzer according to Example of the invention.
  • the supporting substrate is not limited, as long as it is made of a material with desirable magnetic permeability, or has a sufficient magnetically permeable thickness. Particularly preferred are, for example, quartz glass substrates, and silicon substrates.
  • the cover substrate used to seal a solution on the supporting substrate may be made of material, for example, such as inorganic glass and optical polymer that allow the passage of visible light. The wettability of the supporting substrate and the cover substrate should be as high as possible.
  • washed glass is most readily available.
  • Hydrophobic polymers such as PDMS (polydimethylsiloxane) also may be used after a surface hydrophilic treatment performed by introducing an O 2 plasma or carboxyl groups.
  • the substrate wettability is sufficient when it has a contact angle of about 10 to 30° for distilled water. However, the contact angle is more preferably less than 10° for improved effectiveness.
  • a magnetic field generator for attracting magnetic fine particles to the supporting substrate is installed directly below the supporting substrate.
  • the magnetic field generator has a function to switch on and off a magnetic field, or switch magnetic field strengths.
  • the magnetic field generator may use, for example, an electromagnet, a movable permanent magnet, a movable electromagnet, a permanent magnet with a movable magnetic field shield placed between the supporting substrate and the magnet, or an electromagnet.
  • the magnet is selected according to its magnetic force as may be suited for the type of the magnetic fine particles used.
  • a strong magnetic field is needed particularly for immobilization of magnetic fine particles with a particle size of 300 nm or less, and such particles need to be attracted with a surface magnetic flux density of 0.1 T or more for a several seconds. Note here that the required magnetic force varies with the particles size of the magnetic fine particles, the ferrite content, the solvent, and the surface modification on the magnetic fine particles.
  • the term “device” is used to refer to a structure that includes the cover substrate above the supporting substrate, and in which the magnetic field generator is provided on the side of the supporting substrate with a certain space created to seal a solution between the substrates.
  • the device is used by being installed on a movable stage so as to enable a whole scan of a supporting substrate surface.
  • Observation is performed in the following sequence.
  • a reaction liquid containing magnetic fine particles having captured the target biomolecules thereon is placed on the supporting substrate, and the magnetic field generator is turned on to generate a magnetic field.
  • the magnetic field attracts and immobilizes all the magnetic fine particles inside the reaction liquid on the supporting substrate.
  • the magnetic fine particles immobilized on the supporting substrate with the bound fluorescence-labeled biomolecules are exposed to excitation light for imaging. The observed bright spots are then counted to determine the target biomolecule concentration.
  • an immunological analysis method is disclosed in which an analyte antigen prepared is bound to magnetic fine particles that have been conjugated to an antibody against the antigen, and to a fluorescently labeled antibody, and the labeled fluorescent product is detected.
  • the method for producing the capture magnetic fine particles, and the fluorescence labeling method will be described in detail in the Examples below.
  • a device with which analyte biomolecules are two dimensionally spread and immobilized under a magnetic field and a biomolecule analyzer that includes the device and means by which the fluorescence of the fluorescent product is measured.
  • This Example describes the principle of the present invention, examines the principle.
  • a solution 102 is sealed between a pair of parallel flat substrates 101 as shown in FIG. 1 .
  • moving the upper flat substrate 101 with a certain velocity v in x direction moves the fluid along with it in the vicinity of the substrate surface with a flow rate v 1 approximately equal to velocity v (v 1 ⁇ v), whereas the flow rate of the solution 102 approaches 0 toward the surface of the lower, fixed flat substrate 101 .
  • This is due to the generated frictional force between the sealed solution 102 and the flat substrates 101 , and the fluid movement becomes more affected as the wettability of the flat substrates 101 increases.
  • the magnetic fine particles 203 can be evenly immobilized in the form of a film on the substrate surface in a dispersion state, and the fluorescent dye on the substrate can easily be focused. Blocking of excitation light due to trapping of the fluorescent dye by the magnetic fine particles 203 also can be prevented.
  • a device was produced in which one side of the cover glass 302 disposed on a glass slide 301 was lifted up in the manner depicted in FIG. 3 .
  • a solution 304 containing magnetic fine particles 303 is injected into the gap between the two glass plates, and observed from different x-coordinate positions. This enables observation in varying liquid thicknesses of from 0 to 150 ⁇ m with a single substrate. Because the cover glass 302 is tilted, the thickness that appears in one field (430 ⁇ m ⁇ 330 ⁇ m) differs by about 3 ⁇ m on the left and right side of the field. Liquid thickness h was thus determined as the liquid thickness at the field center.
  • the measured bright spot count obtained from the image was divided by the theoretical bright spot count determined from a given number of magnetic fine particles 303 , and plotted against liquid thickness, as shown in FIG. 4 .
  • FIG. 5 represents a graph of average number of bright spot pixels plotted against liquid thickness.
  • the diameters of the magnetic fine particles 303 average number of bright spot pixels
  • increased with decrease in number of bright spots This indicates that the aggregation of the magnetic fine particles 303 had proceeded with increasing liquid thicknesses.
  • Example 8 the following describes an example of procedures of a biomolecule measurement performed with the device that has been adjusted to make the liquid thickness 100 ⁇ m or less from the result described in Example 1.
  • a 26 ⁇ 76 mm glass slide (Matsunami Glass) of 1.2-mm thickness was used as a supporting substrate 801 to immobilize magnetic fine particles in the device.
  • a 0.05-mm thick polyimide tape (Chukoh Chemical Industries) that had been cut into a 1 mm width was then attached to the supporting substrate 801 to provide a square frame of a size that matched the cover member 802 , and the cover member 802 was disposed thereon.
  • a flow tube 804 having a hydrophobic surface such as a silicon tube and a siliconized tip
  • a hydrophobic discharge tube 805 to eject only the air at the outlet.
  • a silicon tube having an outer diameter of 1.0 mm and an inner diameter of 0.5 mm was used. The solution amount is adjusted according to the chamber volume.
  • the solution was used in an amount of about 3 ⁇ l to apply the solution to the whole surfaces of the chamber.
  • 300-nm magnetic fine particles will have a density of 1.1 ⁇ 10 7 /mm 2 when these are laid in a square-grid single layer over the supporting substrate 801 .
  • a single layer of magnetic fine particles can thus be obtained by introducing 3 ⁇ l of a 300 pM magnetic fine particle solution 803 into the chamber.
  • the magnetic field 806 on the device needs to be turned off until the magnetic fine particles become evenly dispersed inside the chamber.
  • the magnetic field 806 may be created by using a permanent magnet or an electromagnet. However, a method that installs and uninstalls a permanent magnet is least expensive and easiest.
  • An electromagnet is convenient to use for switching on and off the magnetic field 806 , but it makes the structure relatively large.
  • a permanent magnet was used to immobilize magnetic fine particles.
  • a neodymium magnet ( ⁇ 20 mm ⁇ 10 mm) with a magnetic flux density of 0.5 tesla (T) was used as the permanent magnet.
  • a holder for fixing the neodymium magnet was disposed immediately below the chamber, and the magnet was manually loaded and unloaded to switch on and off the magnetic field 806 .
  • a method for preparing a sample is described below with reference to FIG. 9 .
  • the biomolecule of interest for detection is an antigen 901
  • the antigen 901 is first captured on magnetic fine particles 903 that have been conjugated with antibody 902 , and labeled with a fluorescent dye 904 that has been conjugated with antibody 902 . All reactions are performed under ordinary temperature with a reaction buffer (tris buffer of pH 8.0, 50 mM NaCl, 0.1% Tween 20).
  • the reaction between the antibody 902 -conjugated magnetic fine particles 903 and the antigen 901 , and the reaction between the fluorescence-labeled antibody 902 and the antigen 901 may be performed in either order, and may be simultaneously performed.
  • the antigen 901 may be any antigen, whereas an antibody with high specificity to the antigen 901 is preferably selected for the antibody 902 .
  • PSA prote specific antigen
  • PSA antibodies need to be provided as antibodies that bind to the magnetic fine particles, and antibodies that are labeled with the fluorescent dye.
  • the antibody 902 may be polyclonal antibody or monoclonal antibody, and the same antibody 902 may be used in the case of polyclonal antibody.
  • the antibody 902 is appropriately selected according to the type of antigen 901 . Desirably,
  • a monoclonal antibody is selected for the magnetic fine particles
  • a polyclonal antibody is selected for the fluorescent dye
  • the antigen 901 is first reacted with the magnetic fine particles 903 that have been conjugated with the monoclonal antibody, and then with the polyclonal antibody.
  • the magnetic fine particles are prepared as particles with a secondary antibody 905 that can bind the antibody 902 while maintaining the activity of the antibody 902 .
  • the magnetic fine particles 903 are readily available from commercial products.
  • anti-mouse IgG-decorated Adembeads may be used as paramagnetic fine particles 903 .
  • the PSA antibodies are mixed and incubated in at least about 10 times the magnetic fine particles decorated with the secondary antibody 905 .
  • the solution is removed, and the conjugates are suspended in a clean buffer. This procedure is repeated until the majority of the unreacted antibodies are removed.
  • Magnetic fine particles decorated with streptavidin also may be used as the magnetic fine particles 903 .
  • streptavidin-decorated Adembeads ( ⁇ 100 nm, ⁇ 200 nm, ⁇ 300 nm) available from Ademtech may be used.
  • the antibody 902 is biotinylated, and bound to the surfaces of the magnetic fine particles 903 .
  • fluorescent dyes are commercially available.
  • Well known examples include FITC, Alexa®, and CY5.
  • FITC fluorescent C
  • Alexa® Alexa®
  • CY5 CY5
  • a high-luminance fluorescent dye having a long quench time examples include a dendrimer-type fluorescent dye in which several hundred molecules of fluorescent dye are bound to a single branching carbon chain; fluorescence polystyrene beads; and quantum dots.
  • fluorescence polystyrene beads are used as fluorescent dye 904 , and fluorescence labeling of antibody 902 with this fluorescent dye is described.
  • the fluorescence polystyrene beads used herein may be one that is available from Invitrogen under the trade name FluoSphere F8771®. These beads are coated with streptavidin, and can bind biotinylated antibody 902 .
  • the antigen 901 is captured between the antibody 902 -conjugated fluorescence polystyrene beads and the antibody 902 -conjugated magnetic fine particles.
  • the antibodies 902 and the magnetic fine particles are introduced into a reaction vessel, and thoroughly stirred therein.
  • the supernatant containing the unreacted antibodies 902 is removed, and the conjugates are suspended in a reaction buffer. Thereafter, a solution with the antigen 901 to be detected is added to the suspension, thoroughly mixed, and incubated for 1 hour.
  • the reaction between antigen 901 and antibody 902 can be accelerated by vertically rotating the reaction vessel, or by shaking and agitating the reaction vessel.
  • the antibody 902 -conjugated magnetic fine particles 903 are mixed in excess of the antigen 901 . Specifically, the antibody 902 -conjugated magnetic fine particles 903 are added in 100 to 10000 times the estimated amount of antigen 901 .
  • the fluorescent dye 904 -conjugated antibodies 902 are then added to the reaction liquid, and incubated for several minutes.
  • the fluorescent dye 904 -conjugated antibodies 902 are also added in excess of the antigen 901 , specifically in 100 to 10000 times, or even in greater amounts with respect to the antigen 901 .
  • the excess addition increases the frequency of collision with the antigen 901 , and improves the capture rate and the fluorescence labeling rate of the antigen 901 .
  • the unreacted fluorescence-labeled antibodies are washed away.
  • the reaction liquid is diluted with washing buffer used in about 5 times the amount of the reaction liquid, and the dilute reaction liquid in the micro tube is inserted into a magnetization magnet holder, and allowed to stand for 2 minutes.
  • the supernatant is completely removed with care that the magnetic fine particles are not aspirated. Thereafter, the same amount of washing buffer is added, and the magnetic fine particles are suspended therein and magnetized. This procedure is repeated about 5 to 7 times to remove the unreacted antibodies 902 conjugated with the fluorescent dye 904 .
  • the liquid is concentrated to an amount to be introduced into the device, and the total amount is injected into the device.
  • the magnetic field 806 on the device is switched off to allow the liquid to evenly spread inside the device. The magnetic field 806 is switched on upon checking that all the liquid has entered the device. In the presence of the magnetic field 806 , formation of film-like magnetic fine particles was confirmed.
  • Example 4 The preferred configuration of a bioanalyzer that includes the device and an incident-light microscope will be described in detail in Example 4 below.
  • the device was placed on an automated stage, and exposed to excitation light through an objective lens 807 from above as shown in FIG. 8 , ( c ). In about 500 fields of scanned image, fluorescence bright spots were observed with hardly any trapping loss by the magnetic fine particles 903 .
  • this Example describes procedures in which the biomolecule of interest for detection is a nucleic acid fragment.
  • a sample nucleic acid fragment 1001 as a detection target is captured on magnetic fine particles 1002 , and labeled with a fluorescent dye 1004 -conjugated nucleic acid fragment 1005 having a sequence 1003 complementary to the sample nucleic acid fragment 1001 .
  • This is a specific hybridization reaction, and was performed in a reaction buffer (PBS buffer of pH 7.4, 50 mM to 1 M NaCl, 0.1% Tween 20).
  • the reaction between the nucleic acid-conjugated magnetic fine particles 1002 and the sample nucleic acid fragment 1001 , and the reaction between the fluorescent dye 1004 -conjugated nucleic acid fragment 1005 and the sample nucleic acid fragment 1001 may be performed in either order, and may be simultaneously performed.
  • the nucleic acid fragment may be a single-strand DNA or RNA. The following specifically describes an example in which microRNA was used as analyte.
  • MicroRNA is a single-stranded nucleic acid fragment of about 20 mer.
  • an adapter nucleic acid sequence 1003 is bound to the 3′ end of the microRNA used as the sample nucleic acid fragment 1001 .
  • a 20-mer poly-A sequence may be used.
  • the complementary sequence fragment 1005 for the adapter is bound to the fluorescent dye 1004 .
  • the fluorescent dye 1004 with the complementary sequence fragment 1005 for the adapter was then mixed with the sample nucleic acid fragment 1001 , and incubated at room temperature for about 1 hour.
  • magnetic fine particles 1002 were prepared as particles with a complementary sequence fragment 1003 for the sample nucleic acid fragment 1001 , and mixed with the sample nucleic acid fragment 1001 previously reacted with the fluorescent dye 1004 . The mixture was then incubated at room temperature for about 1 hour in the same manner as above.
  • Quantum dots are semiconductor fine particles with diameters of several nanometers to several ten nanometers. Quantum dots have a longer lifetime, and are brighter than conventional fluorescent dyes, and different particle sizes fluoresce in different wavelengths.
  • Various types of quantum dots are commercially available, and some are decorated with various functional groups. For example, Invitrogen Qdot 655 Streptavidin®, capable of binding any antibodies may be used as quantum dots, and may be bound to a biotinylated labeled nucleic acid fragment.
  • the streptavidin-decorated Qdot® available from Invitrogen may be used.
  • the binding between the quantum dots and the nucleic acid fragments may be achieved by incubating the mixture of these for at least 30 minutes.
  • the unreacted nucleic acid fragments are removed by using a spin column with a cutoff of 50 kDa after the reaction.
  • the magnetic fine particles with the capture sequence fragment, the fluorescent dye-labeled nucleic acid fragment, and the microRNA adjusted to a concentration of 1 to 100 pM were mixed after being prepared in the manner described above, and the mixture was incubated for 6 hours after being thoroughly stirred.
  • the hybridization reaction can be accelerated by vertically rotating the reaction vessel, or by shaking and agitating the reaction vessel.
  • the magnetic fine particles are mixed in excess of the target nucleic acid fragments. Specifically, the magnetic fine particles were added in 100 to 10000 times the estimated amount of the sample in terms of the number of molecules.
  • the reaction liquid prepared as above was placed on a supporting substrate 1006 , and observed under the attractive force of a magnetic field 1007 as in Example 2. It was found that the total number of bright spots in each sample was proportional to the concentration of the reacted microRNA. Further, with the diameter ⁇ of 100 nm, it was possible to immobilize the magnetic fine particles in a density as high as about 9 times that observed in Example 2 in which the same number of magnetic fine particles was used. Accordingly, the detection time improved by a factor of about 9.
  • the biomolecule analyzer of this Example includes the device that attracts and retains magnetic fine particles on a supporting substrate 1101 under a magnetic field.
  • the device is provided as an integral unit with means to illuminate the supporting substrate 1101 with light, means to supply an analyte biomolecule solution, means to measure fluorescence, and means to operate the supporting substrate.
  • a light microscope with a movable stage 1102 was used.
  • a magnet holder 1103 was placed on the movable stage 1102 , and a device 1104 was fixed thereon.
  • the device is joined to a flow tube 1105 and a discharge tube 1106 in advance.
  • a silicon tube was used as the flow tube 1105 .
  • a magnetic field generator 1107 was used to generate a magnetic field, and attract and immobilize the magnetic fine particles on the surface of the supporting substrate 1103 .
  • An excitation light source 1108 is appropriately selected according to the type of the fluorescent product used. For example, a mercury lamp was used as the light source 1108 when quantum dots were used as the fluorescent dye for fluorescence labeling.
  • the excitation filter 1109 and the excitation light from the excitation light source 1108 travel through a lens 1110 , and are guided into an objective lens 1112 off a dichroic mirror 1111 to illuminate the supporting substrate 1101 .
  • the fluorescence that generates from the fluorescence-labeled molecules on the supporting substrate 1107 propagates in the same light path as the excitation light in the opposite direction, and collected through the objective lens 1112 .
  • the light then passes the dichroic mirror 1111 , and forms an image on the light-sensitive surface of a two-dimensional CCD camera 1114 through an imaging lens 1113 .
  • the scattered rays of the excitation light are removed by an absorbing filter 1115 .
  • the observable bright spots need to be increased to improve the quality of quantification. This can be achieved by moving the movable stage 1103 at high speed, and scanning the whole surface of the supporting substrate 1101 in shorter time periods. It was possible to scan an area of 100 fields (16 mm 2 ) in about 3 minutes with the biomolecule analyzer built from the 20 times objective lens 1112 , a flow pump 1116 , the excitation light source 1108 , the fluorescence detecting unit, the magnetic field generator 1107 , and the movable stage 1103 . This rate is equivalent of observing 1.8 ⁇ 10 7 magnetic fine particles in 3 minutes in a single layer of magnetic fine particles.

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